Chapter 4 —— 42 —— and alignment between donor and target sequences. Of notice, when compared to MMEJ and NHEJ donors, HR and HMEJ donors mitigate indels at chromosomal-recombinant DNA junctions yielding more accurate and directional gene knock-ins [4,6,8]. Multiple physical and chemical transfection methods such as those based on electroporation and polycations, respectively, permit introducing genome editing reagents (e.g. engineered CRISPR nucleases and donor DNA constructs) into human cells. Yet, achieving optimal transfection efficiencies without evident cytotoxic effects is challenging. Moreover, effective transfection of multiple cell types often requires different reagents whose assembly or compositions are sometimes unknown due to proprietary reasons. Transfections are also dependent on systematic optimization of cell typespecific protocols whose ultimate performance often varies as a function of subtle experimental conditions, e.g. distribution of cell-cycle stages in cell populations at the time of transfection. Instead, viral vector transductions display high reproducibility and can be applied in a straightforward manner to different cell types. These features derive from the exquisite finely tuned mechanisms evolved by the vector parental viruses for the delivery of their nuclei acid genomes into host-cell nuclei. Adenoviral (AdV) and adenoassociated viral (AAV) vectors are particularly effective delivery vehicles in a wide variety of mammalian cell types independently of their cell-cycle statuses [10]. AdVs contain a linear protein-capped double-stranded DNA genome (up to 36 kb) [11]; AAVs have a linear single-stranded DNA genome (up to 4.7 kb) with T-shaped hairpin inverted terminal repeats (ITRs) [12]. Previous experiments have demonstrated that, in contrast to free-ended linear DNA, capped double-stranded DNA molecules, including AdV genomes, are refractory to concatemerization and off-target chromosomal insertion [13,14]. Amongst constructs encoding edits-of-interest or programmable nucleases it is especially critical to avoid chromosomal integration of the latter to minimize the potential build-up of off-target effects. Moreover, diversely from AAVs, AdVs can package complete CRISPR-Cas9 constructs encoding
RkJQdWJsaXNoZXIy MTk4NDMw